Treatment modalities for brain and neurological diseases are extremely limited due to the impermeability of the brain's blood vessels to most substances carried in the blood stream. The blood vessels of the brain, referred to collectively as the blood-brain barrier (BBB), are unique when compared to the blood vessels found in the periphery of the body. Tight apposition of BBB endothelial cells (EC) to neural cells like astrocytes, pericytes and neurons induces phenotypic features that contribute to the observed impermeability. Tight junctions between ECs comprising the BBB limit paracellular transport, while the lack of pinocytotic vesicles and fenestrae limit non-specific transcellular transport. These factors combine to restrict molecular flux from the blood to the brain to those molecules that are less than 500 daltons and also lipophilic. Thus, using the large mass transfer surface area (over 21 m2 from 400 miles of capillaries in human brain) of the bloodstream as a delivery vehicle is largely infeasible except in those circumstances where a drug with the desired pharmacological properties fortuitously possesses the size and lipophilicity attributes allowing it to pass freely through the blood vessel. Because of such restrictions, it has been estimated that greater than 98% of all small molecule pharmaceuticals and nearly 100% of the emerging class of protein and gene therapeutics do not cross the BBB.
In addition to the physical barrier presented by the BBB, efflux transporters such as p-glycoprotein (MDR1) and members of the multi-drug resistance-associated protein family (MRP) serve to further limit brain uptake of even those small molecules that are small and lipophilic. FIG. 1A is a micrograph of a section of rat brain (V=ventricle) illustrating the sequestration of horseradish peroxidase in vessels, while in small brain regions perfused by capillaries lacking the BBB, the protein diffuses readily into brain tissue. FIG. 2A also illustrates the impermeable nature of the BBB: histamine (111 Da) remains sequestered within the blood vessels and does not enter the brain interior.
Although neurological diseases such as brain cancer, Alzheimer's disease, Parkinson's disease, and stroke continue to afflict people worldwide, there has been a paucity of new therapies to treat such diseases. Lack of treatment modalities can, in part, be attributed to the lack of effective brain delivery strategies. Due to this lack of treatment methods the National Institutes of Health tumor and stroke progress review groups have even identified the search for innovative strategies for drug or gene targeting through the blood-brain barrier as a top research priority. The National Institutes of Health (NIH) guidelines instruct that such breakthroughs in basic neuroscience can be delivered to the clinic and “require an agent delivery strategy and/or the ability to target specific areas of the brain”. Thus, in the absence of appropriate vehicles for targeting and trans-BBB transport, the pipeline of new CNS medicines will likely continue to be inadequate for the people suffering from neurological disorders. As examples, protein therapeutics known as neurotrophins have been investigated recently for their protective capacity in stroke, reversal of Parkinson's disease symptoms after direct infusion into the brains of human subjects, and for their ability to direct specialized differentiation of neural stem cells for potential treatment of Parkinson's disease and other neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and multiple Sclerosis. Although extremely promising therapeutics, these trophic factors do not readily cross the BBB and will require a noninvasive delivery system for widespread, effective administration.
Present brain delivery strategies are particularly invasive and require circumvention of the BBB. Strategies requiring neurosurgery are used for implantation of polymer particles infused with drug, but the treatment volume is limited because the extracellular fluid in brain tissue is quiescent, and simple molecular diffusion only allows for a modest penetration distance of 2-3 mm. A similar method is direct injection of a drug into the brain ventricles, but the penetration into brain tissue is also limited for such intraventricular injections because the cerebral spinal fluid is rapidly cleared and is turned over 4-5 times daily. In addition, protein therapeutics, such as, for example, Glial-cell Line-Derived, Neurotrophic Factor (GDNF) for Parkinson's disease have been delivered through neurosurgically implanted catheters with continual drug infusion by a peripheral pump. Disruption of the BBB has been investigated using hyperosmolar solutions and vasoactive agents like serotonin and bradykinin peptides to allow free passage of molecules from the blood to the brain. This method is primarily used clinically only in terminal patients because alterations in the BBB can lead to toxic effects due to the free access of solutes and immune factors that are normally excluded from brain.
The aforementioned invasive strategies can have success for those diseases with limited treatment volumes, such as, for localized, non-metastatic brain tumors. However, for chronic conditions requiring a repetitive treatment regimen, or for those diseases present in large portions of the brain such as Alzheimer's disease, a noninvasive drug delivery strategy would be substantially more preferable and practical.
A variety of noninvasive brain drug delivery methods have been investigated that make use of the brain blood vessel network to gain widespread drug distribution. The brain capillary network has an average spacing of just 40 microns between capillaries and is sufficiently dense that each brain cell essentially has its own vessel for nutrient supply (FIG. 2). In addition, if the endothelial barrier of the BBB can be overcome such that a drug is deposited on the brain side of the BBB, the diffusion distance is short enough that each brain cell should be accessible to the drug. As a result, in contrast to invasive methods, a comprehensive treatment volume can result. These noninvasive transport systems/mechanisms can be generally clustered into three groups: 1, non-specific uptake; 2, carrier-mediated transport; and 3, receptor-mediated transport (FIG. 3).
Non-specific uptake mechanisms, while allowing some transport across the BBB, lack sensitivity and specificity. Cationic protein transduction domains fall into the realm of non-specific carriers, and although the HIV TAT peptide was shown to gain access to the brain interstitium after intraperitoneal injection, subsequent pharmacokinetic analysis indicated that the rapid clearance and broad organ uptake would necessitate very high doses to gain a pharmacologic effect. Another non-specific uptake mechanism is the surfactant coating of nanoparticles with polysorbate 80. Although the mechanism of brain uptake is still unresolved, the labile nature of the particles in vivo leads to short-lived pharmacologic effects and possible BBB permeabilization. Both of these non-specific methods suffer from a lack of selective targeting and result in widespread distribution of the active compound throughout the body with concomitant systemic effects.
Carrier-mediated drug transport relies on the presence of endogenous transmembrane proteins that are selective and stereospecific for small molecule solutes. For instance, L-dopa, administered to treat Parkinson's disease gains entry to the brain by utilizing the large amino acid transporter (LAT-1). The successful transport through the blood-brain barrier is a result of L-dopa mimicking the structure of phenylalanine with only the substitution of two hydroxyl groups on the aromatic ring of phenylalanine. Utilization of the saturable biotin transport system for delivery of biotinylated drugs has also been attempted. In addition, it is likely that the efflux of the AIDS drug AZT progresses in a carrier-mediated fashion. However, due to the stereospecificity and steric constraints imposed by these selective membrane pores, applications are potentially limited.
Receptor-mediated transport involves the binding of an exofacial epitope of a cell surface receptor and triggering of an energy intensive transcellular transport process known as transcytosis (FIG. 3). Drugs can be delivered using these portals if conjugated to the natural ligand or an antibody that can trigger the transcytosis process. This method has been successful in allowing for non-invasive transport of small molecules, proteins, genes, nanoparticles, and liposomes up to 100 nm in size. The receptors that are commonly targeted for transcytosis are the low density lipoprotein (LDL) receptor, the transferrin receptor, and the insulin receptor. Similar less specific processes involving absorptive-mediated transcytosis have been used with cationized proteins that promote receptor clustering and activation of the transcytosis pathway. Strategies oftentimes target the cell surface receptor in ways that do not disrupt the normal transport of endogenous ligand. Therefore the impact on normal metabolic pathways is limited. In addition, since transcytosis employs the vesicular trafficking system, this strategy is not nearly as limited by the size and shape constraints of carrier-mediated transport.
Antibodies are particularly well suited for targeting BBB receptor-mediated transcytosis systems given their high affinity and specificity for their ligands. As examples, appropriately-targeted antibodies that recognize extracellular epitopes of the insulin and transferrin receptors can act as artificial transporter substrates that are effectively transported across the BBB and deposited into the brain interstitium via the transendothelial route. Additionally, when conjugated to drugs or drug carriers of various size and composition, the BBB targeting antibodies mediate brain uptake of the therapeutic cargo. Noninvasive transport of small molecules such as methotrexate has been achieved using anti-transferrin receptor antibodies. Proteins such as nerve growth factor, brain derived neurotrophic factor, and basic fibroblast growth factor were delivered to the brain after intravenous administration by using an anti-transferrin receptor antibody. The latter two cases promoted reduction in stroke volume in rat middle cerebral artery occlusion models. Liposomes and liposomes containing genes have also been delivered to the brain in vivo using anti-transferrin receptor antibodies. In particular, gene-containing antibody-targeted liposomes have been targeted to rat brain for restoration of tyrosine hydroxylase activity in an experimental Parkinson's disease model and to primate brain using a humanized anti-insulin receptor antibody. In addition, brain delivery of the new class of RNA interference drugs via pegylated immunoliposomes has been demonstrated to increase survival of mice implanted with an experimental human brain tumor model. Further, anti-transferrin receptor conjugated nanoparticles have been produced. Finally, even if an antibody binds to the brain microvsculature or internalizes without full transcytosis, it can have drug delivery benefits. When conjugated to a liposome or nanoparticle loaded with lipophilic small molecule drugs, it might be possible to raise the local BBB concentration of drug and help circumvent brain efflux systems, thereby facilitating brain uptake. Finally, in the event binding occurs without transcytosis or even internalization, the identification of BBB-specific antibody receptors or ligands will further help to characterize and identify components of the transporter system and help to further optimize antibodies that do internalize and trancytose. Taken together, these results indicate the potential utility of antibody-targeted transcytosis systems for noninvasive trafficking of drugs into the brain.
Regardless of the promise shown for antibody mediated transport in prior studies, the current antibody targeting reagents lack specificity and transport efficiency. Although early results derived from the receptor-mediated transcytosis process are promising due to its robustness in delivery of drugs or drug carriers in many formats, it also has some serious drawbacks that need to be addressed for general clinical success. The present methods rely on receptors that are ubiquitously expressed like the transferrin and insulin receptors. This leads to mis-targeting of potentially expensive drugs that also may have unwanted side effects in tissues other than the brain. In addition, the present methodologies generally result in a low fraction (1-4%) of the injected dose actually reaching the brain target as a consequence of poor targeting and nonideal BBB permeability. This loss of between 96-99% of the administered therapeutic could hamper the development of these delivery approaches given the cost of drug manufacture, especially for protein and gene-based medicines that currently comprise nearly 700 drugs in various stages of clinical trials. Finally, the antibodies used in the aforementioned proof-of-concept experiments are either of murine origin or partially humanized, and this could lead to unwanted immunogenic reactions in human patients. Therefore, the identification of fully human antibodies that specifically recognize brain endothelial receptors would vastly improve the targeting and efficiency of drug delivery while minimizing side-effects.